Infliximab Induction Lacks Efficacy and Increases BK Virus Infection in Deceased Donor Kidney Transplant Recipients: Results of the CTOT-19 Trial.

BACKGROUND: Ischemia-reperfusion (IR) of a kidney transplant (KTx) upregulates TNF α production that amplifies allograft inflammation and may negatively affect transplant outcomes. METHODS: We tested the effects of blocking TNF peri-KTx via a randomized, double-blind, placebo-controlled, 15-center, phase 2 clinical trial. A total of 225 primary transplant recipients of deceased-donor kidneys (KTx; 38.2% Black/African American, 44% White) were randomized to receive intravenous infliximab (IFX) 3 mg/kg or saline placebo (PLBO) initiated before kidney reperfusion. All patients received rabbit anti-thymocyte globulin induction and maintenance immunosuppression (IS) with tacrolimus, mycophenolate mofetil, and prednisone. The primary end point was the difference between groups in mean 24-month eGFR. RESULTS: There was no difference in the primary end point of 24-month eGFR between IFX (52.45 ml/min per 1.73 m 2 ; 95% CI, 48.38 to 56.52) versus PLBO (57.35 ml/min per 1.73 m 2 ; 95% CI, 53.18 to 61.52; P =0.1). There were no significant differences between groups in rates of delayed graft function, biopsy-proven acute rejection (BPAR), development of de novo donor-specific antibodies, or graft loss/death. Immunosuppression did not differ, and day 7 post-KTx plasma analyses showed approximately ten-fold lower TNF ( P <0.001) in IFX versus PLBO. BK viremia requiring IS change occurred more frequently in IFX (28.9%) versus PLBO (13.4%; P =0.004), with a strong trend toward higher rates of BKV nephropathy in IFX (13.3%) versus PLBO (4.9%; P =0.06). CONCLUSIONS: IFX induction therapy does not benefit recipients of kidney transplants from deceased donors on this IS regimen. Because the intervention unexpectedly increased rates of BK virus infections, our findings underscore the complexities of targeting peritransplant inflammation as a strategy to improve KTx outcomes.Clinical Trial registry name and registration number:clinicaltrials.gov (NCT02495077).

Prostate Cancer, Kidney Transplant Wait Time, and Mortality in Maintenance Dialysis Patients: A Cohort Study Using Linked United States Renal Data System Data.

RATIONALE & OBJECTIVE: The impact of prostate cancer on mortality in patients with end-stage kidney disease may be different from the general population. Prostate cancer may also delay the kidney transplant but has not been studied in a population-based cohort. We examined how prostate cancer influenced time to kidney transplant and death in a dialysis population. STUDY DESIGN: Retrospective population-based, risk-set propensity score-matched cohort study. SETTING & PARTICIPANTS: Men, 40-79 years old, who were dialysis-dependent Medicare beneficiaries without prior documented prostate cancer, from the United States Renal Data System. EXPOSURES: Incident prostate cancer, identified using International Classification of Disease, Ninth Revision, Clinical Modification system diagnosis code 185. OUTCOMES: Time to kidney transplant and death. ANALYTICAL APPROACH: Propensity-based risk-set matching to reduce bias between cases and controls. Cox proportional hazards model for time to death, and Fine-Gray competing risk model for time to kidney transplant. RESULTS: Among a total of 588,478 male dialysis patients who met the eligibility criteria, 18,162 had claims for prostate cancer. After propensity-based risk-set matching, 15,554 pairs of prostate cancer cases and controls were identified. Among the matched pairs, survival rates were 76%, 48%, and 30% at 1, 3, and 5 years in the prostate cancer group, compared with 80%, 51%, and 33% in the control group, with relative mortality of 95%, 94%, and 91% respectively (log-rank test P < 0.001). Prostate cancer was associated with a 22% lower likelihood of kidney transplant (HR: 0.78; 95% CI: 0.72-0.85) and 11% higher likelihood of death (HR: 1.11; 95% CI: 1.08-1.14) compared with controls. Kidney transplant was associated with a 4-fold improvement in overall survival, both in patients with and without prostate cancer (HR: 0.20; 95% CI: 0.18-0.21). LIMITATIONS: Retrospective registry study. CONCLUSIONS: Prostate cancer is associated with a modest increase in the risk of death and time to transplant in patients with end-stage kidney disease. Kidney transplant is associated with the same degree of survival benefit among those with pretransplant prostate cancer as those without.

Securing the future of kidney transplantation by addressing the challenges of transplant nephrology.

Kidney transplant is a life-changing procedure, and transplant nephrologists, as part of a larger transplant team, play an important role in the field by managing the complex medical needs of transplant patients. The subspecialty of transplant nephrology, however, faces structural challenges related to its workforce, reporting structures, compensation, research and innovation, and health care information technology. The position of transplant nephrology at the academic and operational intersection of medicine and surgery may limit its access to critical resources, hinder academic promotion, and contribute to physician burnout. The authors provide an overview of the subspecialty transplant nephrology and propose solutions. Collaborative efforts that fortify the subspecialty of transplant nephrology will ultimately improve the lives of patients suffering from kidney disease.

Kidney disease in Uganda: a community based study.

BACKGROUND: Chronic kidney disease (CKD) is a major cause of morbidity and mortality in Sub-Saharan Africa (SSA). The majority of studies on CKD in SSA have been conducted among HIV-infected populations and mainly from large health facilities. We determined the prevalence of CKD and its predictors among populations in communities in central Uganda. METHODS: A cross-sectional study was conducted in Wakiso district using multi-stage sampling. Data was collected on age, sex, socio-economic status, history of alcohol intake, diabetes mellitus, hypertension and smoking. Measurement of blood pressure, weight and height to determine body mass index (BMI) and investigations including HIV testing, fasting blood sugar, creatinine and urinalysis were conducted. Logistic regression was used to estimate the strength of the association between variables and the presence of CKD estimated using the Cockcroft Gault formula. RESULTS: A total of 955 participants aged 18-87 years were enrolled into the study. The median age was 31 years (Interquartile range 24-42) and majority (67%) were female. Up to 21.4% (204/955) had abnormal renal function with CKD stage 1 in 6.2% (59/955), stage 2 in 12.7% (121/955), stage 3 in 2.4% (23/955), CKD stage 4 in 0% and CKD stage 5 in 0.1% (1/995). Female gender OR 1.8 (95% Confidence Interval [CI] 1.2-2.8), age >30 years OR 2.2(95% CI 1.2-3.8) and high social economic status OR 2.1 (95% CI 1.3-3.6) were associated with increased risk of CKD while BMI > 25Kg/m2 was protective against CKD OR 0.1 (95% CI 0.04-0.2). Traditional risk factors such as HIV-infection, diabetes mellitus, smoking and alcohol intake were not found to be significantly associated with CKD. CONCLUSION: We found a high prevalence of kidney disease in central Uganda. Interestingly the traditional risk factors associated with CKD previously documented, were not associated with CKD.

Costimulatory Blockade and Use of Mammalian Target of Rapamycin Inhibitors: Avoiding Injury Part 1.

Although calcineurin inhibitor drugs have been the mostly used therapy in modern immunosuppression in kidney transplantation, their effect on kidney allograft dysfunction has been suboptimal as far as preservation of kidney function is concerned. Additionally, there are metabolic and other nonmetabolic effects including increased risk of malignancy that has necessitated the use of mammalian target of rapamycin inhibitors to reduce exposure to calcineurin inhibitors. Mammalian target of rapamycin inhibitors, both sirolimus and everolimus, have been studied in several trials to facilitate preservation of kidney function with variable effects on kidney allograft function and immunogenicity. Preservation of kidney function is increasingly becoming the mainstay of immunosuppression not only in kidney transplantation, but also in extrakidney transplantation. The best kidney outcomes have been reported in calcineurin inhibitor withdrawal studies using mammalian target of rapamycin inhibitors, in kidney transplant recipients with stable kidney function. This review article summarizes data from several studies in which mammalian target of rapamycin inhibitors have been used to reduce exposure to or withdraw calcineurin inhibitors in an attempt to preserve kidney function.

Adverse Outcomes of Tacrolimus Withdrawal in Immune-Quiescent Kidney Transplant Recipients.

Concerns about adverse effects of calcineurin inhibitors (CNIs) have prompted development of protocols that minimize their use. Whereas previous CNI withdrawal trials in heterogeneous cohorts showed unacceptable rates of acute rejection (AR), we hypothesized that we could identify individuals capable of tolerating CNI withdrawal by targeting immunologically quiescent kidney transplant recipients. The Clinical Trials in Organ Transplantation-09 Trial was a randomized, prospective study of nonsensitized primary recipients of living donor kidney transplants. Subjects received rabbit antithymocyte globulin, tacrolimus, mycophenolate mofetil, and prednisone. Six months post-transplantation, subjects without de novo donor-specific antibodies (DSAs), AR, or inflammation at protocol biopsy were randomized to wean off or remain on tacrolimus. The intended primary end point was the change in interstitial fibrosis/tubular atrophy score between implantation and 24-month protocol biopsies. Serially collected urine CXCL9 ELISA results were correlated with outcomes. The study was terminated prematurely because of unacceptable rates of AR (4 of 14) and/or de novo DSAs (5 of 14) in the tacrolimus withdrawal arm. Positive urinary CXCL9 predated clinical detection of AR by a median of 15 days. Analyses showed that >16 HLA-DQ epitope mismatches and pretransplant, peripheral blood, donor-reactive IFN-γ ELISPOT assay results correlated with development of DSAs and/or AR on tacrolimus withdrawal. Although data indicate that urinary CXCL9 monitoring, epitope mismatches, and ELISPOT assays are potentially informative, complete CNI withdrawal must be strongly discouraged in kidney transplant recipients who are receiving standard-of-care immunosuppression, including those who are deemed to be immunologically quiescent on the basis of current clinical and laboratory criteria.

Fever, infection, and rejection after kidney transplant failure.

BACKGROUND: Patients returning to dialysis therapy after renal transplant failure have high morbidity and retransplant rates. After observing frequent hospitalizations with fever after failure, it was hypothesized that maintaining immunosuppression for the failed allograft increases the risk of infection, while weaning immunosuppression can lead to symptomatic rejection mimicking infection. METHODS: One hundred eighty-six patients with failed kidney transplants were analyzed for rates of hospitalization with fever within 6 months of allograft failure. Patients were stratified by the presence of full immunosuppression versus minimal (low-dose prednisone) or no immunosuppression, before hospital admission. Subsequent rates of documented infection and nephrectomy, as well as patient survival, were ascertained. RESULTS: Hospitalization with fever within 6 months of allograft failure was common, occurring in 44% of patients overall. However, among febrile hospitalized patients who had been weaned off of immunosuppression before admission, only 38% had documented infection. In contrast, 88% of patients maintained on immunosuppression had documented infection (P<0.001). In both groups, dialysis catheter-related infections were the most common infection source. Allograft nephrectomy was performed in 81% of hospitalized patients with no infection, compared to 30% of patients with documented infection (P<0.001). Mortality risk was significantly higher in patients with concurrent pancreas transplants or who were hospitalized with documented infection. CONCLUSIONS: Maintenance immunosuppression after kidney allograft failure was associated with a greater incidence of infection, while weaning of immunosuppression commonly resulted in symptomatic rejection with fever mimicking infection on presentation. Management of the failed allograft should include planning to avoid both infection and sensitizing events.

Urinary-cell mRNA profile and acute cellular rejection in kidney allografts.

BACKGROUND: The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. METHODS: We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS: A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS: A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

The renal transcriptome of db/db mice identifies putative urinary biomarker proteins in patients with type 2 diabetes: a pilot study.

We sought to identify novel urinary biomarkers of kidney function in type 2 diabetes. We screened the renal transcriptome of db/db and db/m mice for differentially expressed mRNA transcripts that encode secreted proteins with human orthologs. Whether elevated urine levels of the orthologous proteins correlated with diminished glomerular filtration rate was tested in a cross-sectional study of n = 56 patients with type 2 diabetes. We identified 36 putative biomarker genes in db/db kidneys: 31 upregulated and 5 downregulated. Urinary protein levels of six selected candidates (endothelin-1, lipocalin-2, transforming growth factor-ß, growth and differentiation factor-15, interleukin-6, and macrophage chemoattractant protein-1) were elevated in type 2 diabetic patients with subnormal glomerular filtration rate (i.e., <90 ml·min(-1)·1.73 m(-2)), independent of microalbuminuria, age, sex, race, and use of angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists. In contrast, urinary levels of fibroblast growth factor were not increased. A composite variable of urine albumin and any of the six candidate markers was associated with subnormal estimated glomerular filtration rate more closely than albumin alone. In addition, urinary endothelin-1, growth and differentiation factor-15, and interleukin-6 were associated with a marker of proximal tubule damage, N-acetyl-ß-d-glucosaminidase activity. These results suggest that gene expression profiling in diabetic mouse kidney can complement existing proteomic-based approaches for renal biomarker discovery in humans.

Donor phosphorus levels and recipient outcomes in living-donor kidney transplantation.

BACKGROUND AND OBJECTIVES: In living-donor kidney transplantation, various donor factors, including gender, age, and baseline kidney function, predict allograft function and recipient outcomes after transplantation. Because higher phosphorus is predictive of vascular injury in healthy adults, the effect of donor phosphorus levels on recipient renal function after transplantation was investigated. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Phosphorus levels in 241 living donors were analyzed from a 7-year period, and recipient renal function and acute rejection at 1 year posttransplantation were examined controlling for other influencing factors, including multiple donor variables, HLA matching, and acute rejection. RESULTS: Female and African-American donors had significantly higher phosphorus levels predonation. By multivariable analysis, higher donor phosphorus correlated with higher recipient serum creatinine (slope=0.087, 95% confidence interval [CI]: 0.004 to 0.169, P=0.041) and lower recipient estimated GFR (slope=-4.321, 95% CI: -8.165 to -0.476, P=0.028) at 12 months. Higher donor phosphorus also displayed an independent correlation with biopsy-proven acute rejection and delayed or slow graft function after transplantation. CONCLUSIONS: In a cohort of living kidney donors, higher donor phosphorus correlated with female gender and African-American ethnicity and was an independent risk factor for early allograft dysfunction after living-donor kidney transplantation.

Induction antibody therapy in kidney transplantation.

Antilymphocyte antibodies have been used for the prevention or treatment of acute rejection in kidney transplant recipients since the 1960s. Both monoclonal and polyclonal agents now are available and generally are classified as either lymphocyte-depleting or nondepleting agents. Use of such antibodies for induction therapy in the immediate postoperative period has varied over the years. Currently, induction antibodies are administered to more than 70% of kidney transplant recipients in the United States. However, the choice of specific agents and the patients for whom they are used vary substantially between and within transplant centers. Many centers use antibody induction therapy only in patients perceived to be at high risk of acute rejection or delayed graft function. Recently, induction antibody therapy also has become the standard of practice in protocols designed to facilitate minimization of such maintenance immunosuppressive drugs as corticosteroids or calcineurin inhibitors. The benefits of induction therapy, including a decreased incidence and delayed onset of acute rejection, must be balanced against the considerable cost and side effects of the individual agents, including risk of infection. Some, but not all, antibodies are associated with increased risk of posttransplantation lymphoproliferative disease and other malignancies.

Management of the kidney transplant recipient.

The short-term outcomes of kidney transplant recipients have improved dramatically in the past 20 years, in large part resulting from the availability of more potent immunosuppressive drugs capable of preventing or treating acute allograft rejection. Ironically, side effects from these same immunosuppressants play a role in the long-term morbidity and mortality of this patient population. As kidney transplant recipients survive for longer periods of time with functioning allografts, primary care physicians will likely become more involved in their management, mandating at least a basic understanding of immunosuppression and its complications.

Comparing early withdrawal or avoidance of steroids with standard steroid therapy in kidney transplant recipients.

This Practice Point commentary discusses the findings and limitations of an open-label, multicenter, prospective trial conducted by Vincenti et al., in which kidney transplant recipients receiving basiliximab, ciclosporin microemulsion, and enteric-coated mycophenolate sodium were randomized to standard corticosteroid therapy, steroid withdrawal 7 days after transplantation, or complete steroid avoidance. The trial failed to show noninferiority of the steroid-sparing arms in terms of calculated glomerular filtration rate at 12 months. In addition, the steroid-sparing groups had an increased cumulative incidence of biopsy-proven acute rejection at 12 months. This commentary highlights the issues to consider when interpreting and generalizing these results, including the under-representation of African Americans in the study population, the short duration of follow-up, and the switching of some patients between steroid-containing and steroid-sparing immunosuppressive regimens. The benefits of steroid-free immunosuppression versus low maintenance doses of steroids in kidney transplant recipients remain unclear.

Use of sirolimus in solid organ transplantation.

Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that inhibits cell cycle progression and has proven to be a potent immunosuppressive agent for use in solid organ transplant recipients. The drug was initially studied as an adjunct to ciclosporin (cyclosporine) to prevent acute rejection in kidney transplant recipients. Subsequent studies have shown efficacy when combined with a variety of other immunosuppressive agents. The most common adverse effects of sirolimus are hyperlipidaemia and myelosuppression. The drug has unique antiatherogenic and antineoplastic properties, and may promote immunological tolerance and reduce the incidence of chronic allograft nephropathy. Although sirolimus is relatively non-nephrotoxic when administered as monotherapy, it pharmacodynamically enhances the toxicity of calcineurin inhibitors. Ironically, the drug has been used to facilitate calcineurin inhibitor-free protocols designed to preserve renal function after solid organ transplantation. Whether sirolimus can be used safely over the long term with low doses of calcineurin inhibitors requires further study. The use of sirolimus as a corticosteroid-sparing agent also remains to be proven in controlled trials. Postmarketing studies have revealed a number of unforeseen adverse effects including impaired wound healing and possibly proteinuria, oedema, pneumonitis and thrombotic microangiopathy. Overall, sirolimus is a powerful agent when used judiciously with other available immunosuppressants. As is true for all immunosuppressive drugs available for treatment of solid organ transplant recipients, the efficacy of the drug must be balanced against its considerable adverse effects.

Heterogeneous alterations in human alloimmunity associated with immunization.

BACKGROUND: The presence of alloantibodies and/or alloreactive T cells in a patient prior to a transplant can impact graft outcome. Environmental factors, including therapeutic vaccinations, may influence the strength and/or specificity of alloimmunity. METHODS: To address this issue, we prospectively evaluated the effects of two different immunization protocols in human subjects on cellular alloimmunity using an IFNgamma ELISPOT assay and on alloantibody reactivity by flow cytometric analysis of HLA-coated beads. RESULTS: Vaccination/immunization was associated with augmentation of cellular and/or humoral alloimmune reactivity in >50% of the test subjects. The effects were heterogeneous in that some detected increases were transient, peaking 30-60 days postimmunization, whereas others persisted for the length of the study. Antibodies reactive to the immunizing agent did not cross react with the detected alloantibodies, suggesting that the augmentation of alloimmune reactivity was most likely due to a nonspecific adjuvant effect from the vaccine. CONCLUSIONS: Therapeutic vaccinations can alter the strength of cellular and humoral alloimmunity in humans. The results suggest that serial immune monitoring of alloreactivity might be beneficial when immunizations are administered to potential transplant recipients.

Pre-transplant IFN-gamma ELISPOTs are associated with post-transplant renal function in African American renal transplant recipients.

Final crossmatch testing is routinely used to assess the risk of antibody-mediated graft injury/rejection post-transplant. Analogously, we postulated that quantitative measurements of anti-donor effector/memory T cells pre-transplant would independently assess post-transplant risk. To address this hypothesis, we determined the frequencies of pre-transplant, donor-specific interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spots (ELISPOTs) and correlated the results with post-transplant outcomes in 37 African American recipients of deceased donor kidney transplants treated with tacrolimus- and sirolimus-based immunosuppression. A positive ELISPOT test (>25 spots/300,000 cells) was detected in 14 (38%) of 37 patients. The incidence of biopsy-proven acute rejection was 50% (7/14) in ELISPOT-positive versus 17% (4/23) in ELISPOT-negative patients (p=0.036). Calculated glomerular filtration rate (MDRD) at 12 months was 37+/-16 mL/min in ELISPOT-positive versus 55+/-20 mL/min in ELISPOT-negative patients (p=0.01). ELISPOT status remained a correlate of allograft function at 12 months by linear regression analysis (p=0.001), independent of rejection and other contributing variables. Pre-transplant donor-directed IFN-gamma ELISPOT assessment of anti-donor cellular immunity may function as a 'cellular crossmatch' and independently correlates with renal allograft function in African Americans receiving tacrolimus- and sirolimus-based immunosuppression.

A multicenter pilot study of early (4-day) steroid cessation in renal transplant recipients under simulect, tacrolimus and sirolimus.

This study presents the first prospective multicenter study assessing sirolimus-based immunosuppression with early (4-day) corticosteroid withdrawal (CSWD) in renal transplantation. Immunosuppression included: anti-IL-2 receptor antibody and tacrolimus/sirolimus. Inclusion criteria included adult primary recipients. Exclusion criteria included: (i) African Americans, (ii) current PRA >50%, (iii) multiple organ transplants, (iv) WBC < 3000 cells/microL and (v) fasting hypercholesterolemia/hypertriglyceridemia. The primary endpoints were acute rejection and the proportion of patients off corticosteroids. Seventy-seven patients were enrolled: mean age of 49.7 +/- 12 years. Transplants included: cadaveric (26%) and living donor (74%). Patient and graft survival were 100%. Biopsy proven acute rejection occurred in 13%; presumptive rejection in 10.5%. Banff grades included: IA (seven patients), IB (one patient), IIA (one patient) and IIB (one patient). Renal function at 1 year: serum creatinine (1.18 +/- 0.06 mg/dL). Mean weight gain was minimal at 1 year: 3 +/- 2 kg/patient. Mild increases in total, LDL and HDL cholesterol were observed and new antilipid agent use occurred in 26 patients. In conclusion, early CSWD under tacrolimus/sirolimus-based immunosuppression in selected, low-risk renal transplant recipients provides: (i) excellent patient and graft survival, (ii) good renal function, (iii) reduced hyperlipidemia and antilipid agent use and (iv) low acute rejection rates.

Alloreactivity in renal transplant recipients with and without chronic allograft nephropathy.

The pathogenesis of chronic allograft nephropathy (CAN) involves both immunologic (antigen-dependent) and nonimmunologic (antigen-independent) mechanisms. In order to provide further insight into the immunologic basis of this disease, a cross-sectional analysis of cellular and humoral immunity in human renal allograft recipients with or without deteriorating renal function and biopsy proven CAN was performed. Interferon-gamma enzyme-linked immunosorbent spot assays were used to assess cellular immunity to donor, or fully mismatched third-party stimulator cells (direct pathway), and to synthetic peptides derived from donor HLA molecules (indirect pathway). Anti-HLA antibodies were evaluated by flow cytometry using HLA-coated beads. Both the mean frequencies of donor-reactive peripheral blood lymphocytes and the number of individuals who responded to donor antigens per group were statistically higher in CAN patients versus control subjects (P < 0.02). Calculated ratios of donor/third-party enzyme-linked immunosorbent spot responses showed mean values of 2.61 +/- 3.0 in the CAN group, with ratios of 0.50 to 0.72 +/- 0.42 in control subjects (P < 0.001), confirming that direct, donor-specific cellular immunity predominated in patients with CAN. Fifty percent of CAN patients studied exhibited donor peptide reactivity compared with only 28.6% in control subjects. Finally, 33% of patients in the CAN group developed new posttransplantation anti-HLA antibodies compared with only 4% in the control group (P < 0.05). Overall, the results suggest that persistent cell-mediated and humoral alloimmunity contribute to the development of CAN and further demonstrate that anti-donor immunity in patients with CAN is heterogeneous. Immune monitoring to predict long-term outcome should include multiple measures of cellular and humoral immunity.